[Expression plasmid-host strain using chromosome-plasmid balanced lethal system based on the Escherichia coli thyA]

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• Traditional plasmid vectors often rely on antibiotic resistance genes, which can have drawbacks in therapeutic applications.
• The development of non-antibiotic selection systems is crucial for advancing gene therapy and vaccine vectors.
• The thyA gene offers a potential nutritional marker for developing stable, antibiotic-free genetic systems.

Purpose of the Study:

• To construct and validate a chromosome-plasmid balanced lethal system for DNA vaccine and protein expression.
• To create novel vectors utilizing the thyA gene as a nutritional marker, replacing antibiotic resistance.
• To assess the efficiency, stability, and potential of this system as a gene vaccine vector.

Main Methods:

• Amplification and cloning of thyA genes from E. coli and V. cholerae into a modified pCDNA3 vector.
• Construction of two new plasmids, pcDNATE and pcDNATC, featuring the thyA nutritional marker.
• Transformation into a deltathyA E. coli strain to establish chromosome-plasmid balanced lethal systems.
• Cloning of the DsRed2 reporter gene into the constructed plasmids for expression analysis.
• Transfection into HEK293 cells and analysis of DsRed2-myc expression via fluorescence microscopy and Western blot.
• Assessment of plasmid stability over 20 generations in the absence of thymidine.

Main Results:

• Two functional chromosome-plasmid balanced lethal systems based on the thyA gene were successfully developed.
• Recombinant plasmids pcDNATE-DsRed2 and pcDNATC-DsRed2 facilitated the expression of DsRed2-myc.
• The reporter gene expression was confirmed through fluorescence microscopy and Western blot assays.
• No loss of recombinant plasmids was observed after 20 generations in cultures lacking thymidine.
• The system demonstrated stability comparable to traditional antibiotic-resistant plasmid vectors.

Conclusions:

• The developed chromosome-plasmid balanced lethal system is an effective vector for target gene expression.
• This antibiotic-free system exhibits significant stability, rivaling conventional antibiotic-resistance markers.
• The system holds considerable promise as a gene vaccine vector, overcoming limitations associated with drug resistance markers.